CA2017265C - Cosmetic composition against ageing of the skin - Google Patents

Abstract

The invention relates to a cosmetic composition comprising a mixture of (a) betaine, (b) ATP or an ATP generating system, (c) a magnesium salt, and (d) a potassium salt, in a cosmetically acceptable vehicle for topical administration and to a method for counteracting ageing of the skin in a living human which utilizes the new composition.

Description

The present invention relates to a cosmetic composition for combatting ageing of the skin which comprises an ademe~ionine generating system. The present invention also refers to a method of treating the skin to counteract ageing by applying the new composition thereto.
Ademetionine is the International Non-proprietary Name for the inner salt of (S)-5'-[(3-amino-3-carboxypropyl)methylsulfoniol-S'-desoxyadenosine hydroxide of the formula:

l N

N ~ ~CH2-' -CH2-CH2-CH-COO Q
OH OH
also indicated as S-adenosyl-L-methionine or, more simply, as SAMe.
Ademetionine is a physiological molecule, almost ubiquitously distributed within the organism tissues and fluids wherein it participates in important biological processes as a donor of methyl groups for a number of transmethylation reactions and as a precursor of physiological sulfur compounds such as glutathione, cysteine, taurine, and coenzyme A.
It is known that ademetionine levels are high in the infant and in the adolescent, they diminish in the adult and lower further in the pre-senile and senile age.
It is also known that endogenous ademetionine forms at the cell level starting from methionine by the action of adenosyltransferase, of betaine as donor of methyl groups, and of adenosyltriphosphoric acid (ATP), in the presence of magnesium and potassium ions.
It has also been found (FR-A-2623396, published on May 26, 1989) that ademetionine has a skin anti-ageing action and that it can be employed for cosmetic purposes directly on the skin.
The anti-ageing effect of ademetinnine is due to its biochemical properties. Transmethylation takes place on biological molpcllles~
mainly on skin proteins, nucleic acids and phosrhnlipids which are thereby biotransformed and enter the anabolic and catabolic cycles.

This anabolic-catabolic activity at the epidermal cell level favouræ their regeneration while the action on methyltransferases, by increasing phospholipids methylation, make the cell membranes more fluid, thus slowing down the natural skin ageing process.
This membrane-fluidizing action has been demonstrated on human epidermal cells. The membrane microviscosity of human epidermal cells has been studied by the Shinitzky et al method (J. Biol. Chem. 1974, 249, 2652-2657) using diphenylhexatriene as the fluorescent marker. The presence of ademetionine, at a molar concentration of from 106 to 10-4, reduces membrane viscosity by 10 to 25%.
However, for a cosmetic use, ademetionine has the disadvantage of being unstable and rather unsuitable to be handled in the cosmetic industry.
It has now been found, in invitro tests, that betaine, also at very low concentrations, significantly affects ademetionine levels in human skin fibroblasts.
It has also been found that it is possible to obtain the same effects observed with ademetionine by treating the skin with a mixture of betaine, ATP or an ATP
generating system, a potassium salt and a magnesium salt, in a cosmetically acceptable vehicle, thus generating ademetionine in situ.
In accordance with one aspect of the present invention, there is provided a cosmetic composition which comprises a mixture of:
(a) "betaine", as defined below, (b) ATP or an ATP generating system, (c) a magnesium salt, and (d) a potassium salt, in a cosmetically acceptable vehicle for topical administration. Such mixture is contained in the composition in an amount effective to improve the appearance of ageing skin.

~' As used herein, the term "betaine" encompasses the com~ou.-d of formula (CH3)3~CH2-COO as well as the correspon~i~g addition salts with dermocompatible acids and the hydrated forms thereof.
The term "ATP generating system" or "ATP-GS" refers to any biological extract which is capable of increasing the respiratory cellular activity within the mitho-condria, thus accelerating the cellular metabolism and hence the transformation of the products of the glycolysis and of the Krebs cycle into ATP. By increasing the respiratory cellular activity in the last step of the respiratory chain, ATP is generated immediately and directly via biochemical transformation of glucose in ATP, and then via the intermediate pyruvic acid which in its turn produces ATP (T~h~; nger -Principes biochimiques - Flammarion Medicine Science Ed., 1985, 497-498).
Among said biological extracts, those derived from eucaryotic cells, in particular from yeasts, and obtAi~e~
by conventional extraction and purification pror~Cc~s which maintain the biochemical components of the cells, mainly amino acids, peptides and enzymes, almost intact, are preferred.
These yeast extracts are known in the literature and are also commercially available, e.g. under the trade mark VITACELL LS 1917 (manufactured by the French company Laboratoires Sérobiologiques S.A.).
Also preferred are those biological extracts obtained from bovine spleen, well known in literature and commercially available, e.g. under the trade marks K~vllALINE (manufactured by the Swiss company PENTAPHARM) and OXYDERMINE (manufactured by the French company SEDERMA). Such extracts are also obtained by convention-al extraction and purification pror~ss~C which maintain the biochemical components of the cells almost intact.
As far as the magnesium salt as well as the potassium salt are concerned, any organic or inorganic '~aL

3a 2 0 1 7 2 6 5 salt may suitably be employed provided it is dermocom-patible. Gluconates, pyroglutamates and chlorides are however the preferred anions.
British patent application GB-A-2,151,924 describes a cosmetic or dermatological composition-for skin care which comprises, as essential components, a plant, extract from Oenv~,erd biennis and a spleen extract. While Oenothera oil is employed to create a barrier on the epidermis which controls the loss of water from the skin, the spleen extract is employed to activate the cellular respiratory activity, a well known effect also referred to above.
French patent application FR-A-2,608,393 claims pharmaceutical or cosmetic compositions, or compositions useful as bases for the preparation of pharmaceutical or cosmetic compositions which comprise a nitrogen contain-ing compound such as an amino acid, an oligo- or poly-peptide, a protein or a derivative thereof, including also betaine. According to the teachings of said French patent application, P

20~726~

which however does not describe compositions such as those defined in the present application, said preparations present - depen~;ng on the particular formulation - different activities : hydrating, nourishing, regenerating, protecting and stimulating the growth of epidermal and denmal cells or piliferous bulbs.
The components of the cosmetic preparations of the present invention may suitably be employed therein in the following pLoporLions (the percentages are by weight) :
(a) betaine from 0.001 to 1 %
(b) ATP (or ATP-GS) from 0.045 to 4.5 %
(c) Mg from 10 ppb to 0.3 %
(d) K from 0.0001 to 0.6 %
Preferably, said components are employed in the following proportions (the percentages are by weight) :
(a) betaine from 0.03 to 0.3 %
(b) ATP (or ATP-GS) from 0.15 to 1.5 %
(c) Mg from 15 ppb to 0.1 %
(d) K from QOO1 to 0.2 %
Preferred cosmetic preparations according to the present invention comprise :
(a) betaine from 0.05 to 0.25 %
(b) ATP-GS (such as yeast extracts such as e.g. VITACELL LS
l9l7 or bovine spleen extracts such as REVITALINE or OXYDERMINE) from 0.25 to 1 %
(c) Mg from 20 ppb to 0.06 %
(d) K from Q002 to 0.1 %
For the preparation of the cosmetic compositions according to the present invention, the ~ ~n~nts are mixed with cosmetic excipients in order to prepare creams, lotions, emNlsions or solutions.
In particlllAr, the active components are admixed with the excipients conventionAlly employed in the cosmetic field such as for example, fats of animal or vegetal origin, vegetable oil, fatty acids, alcohols, polyalkylene glycols, waxes, petroleum jellies and polyesters, which can be used in ~CsocjAtion with water and jelling agents provided they are compatible therewith.

.

Other ingredients compatible with the active components, such as preservatives, e.g. 4-hyd~oxybenzoic acid esters, antioxidants, e.g. butylhydroxytoluene or vitamin E derivatives, or fragrances, can be added to these preparations.
S Fatty acids employed as adjuvants in the cosmetic compositions of the present invention may be saturated or unsaturated, may contain from 10 to 22 carbon atoms, be unsubstituted or hydL~y-substituted and in the form of the free acids or of the ~lkAline salts thereof.
The cosmetic compositions according to the present invention can be in the form of a cream wherein the active components are associated with the excipients which are commonly used in cosmetology and which are compatiblè therewith, such as lanolin or its derivatives.
The cosmetic compositions according to the present invention can also be in the form of a gel in a suitable excipient such as a cellu-lose ester, an acrylic polymer, a fatty acid ester, e.g. octyl palmi-tate or stearate, or other gelling agents.
According to another embodiment of the present invention, the above active principles are - at least in part - incorporated into phospholipid vectors, in the form of lamellar phases, spherical vesic-les or hollow cylinders of phospholipid layers alternated with aqueous layers, generally designated as "liposomes". The selection of the phospholipids to be used in the preparation of the liposomes as well as the methods for the preparation and utilisation thereof in the liposomal compositions according to the present invention can readily be carried out by following the literature tea~hings (see for instance "Liposomes" - M. Ostro Ed., Marcel Decker, New York, 1983).
The cosmetic compositions of the present invention can also be in the form of a lotion, a solution or a mi~r. l~ion wherein the active components are dissolved or micro~isr~rsed.
The cosmetic compositions according to the invention can therefore be in the form of a microdispersion of the components in a liquid conta-ining water, oil and one or more surfactants. These dispersions have the same properties as microemulsions and the same appearance as true solutions. They can be prepared immediately before use. These microem-ulsions possess a good stability and can be stored for the time -necessary for their use, at temperatures from 0C to 60C without sedimentation of the constituents or irreversible phase separation.
The surfactants which can suitably be employed in these compositions are selected from those surface-active agents which can be used in 05 cosmetology.
As non-limiting examples there may be cited: sorbitol esters and their polyethoxylated derivatives, polyethoxylated castor oils (hydrogenated or non-hydrogenated), ethylene oxide/propylene oxide block polymers, polyethoxylated fatty alcohols and sterols, sodium laurylsulfate, sodium dioctylsulfosuccinate, egg or soya lecithins, and polyeth-oxylated silicon oils.
The effect of betaine on SAMe formation has been evaluated by the results obtained with betaine increasing concentrations in cultu-res of human skin fibroblasts. More particularly human skin fibroblasts (ATCC No. CRL 1513 - 2.104 cells/ml) have been cultured for 6 days in Dulbecco's Modified Eagle Medium (DMEM - 250 ml containers) containing 20 % of foetal calf serum, in the presence of increasing concentrations (from 10 5M to 5.10 4M)) of betaine. Then, the cells were taken off by treatment with trypsine (0.05 %) and EDTA (0.02 %) and centrifuged. The supernatant was separated, precipitated by the addition of trichloro-acetic acid (0.2N, v/v) and centrifuged again. The supernatant was then recovered and lyophilized. The lyophilized material was taking up in water and analyzed by HPLC (column : ~ucleosyl C8; Eluent: Solvent A
(Sodium acetate 8.2 g, Sodium octylsulfonate 200 mg, Citric acid 4.2 g, EDTA 50 mg and water q.s. to I 1) 95 % and Solvent B (Methanol) 5 %) using an U.V. detector at 260 nm. The amount of SAMe has been determined in comparison with a calibration curve prepared under the same experimental conditions.
Betaine concentration SAMe concentration 30 (10 M) (nMoles/10 cells) 0 0.54 1 0.62 0.94 ~.72 1.90 By using the cosmetic compositions according to the present invention, which contain, in addition to betaine, ATP or an ATP gener-ating system, a magnesium salt, and a potassium salt, after 30 days of regular application, a notable improvement in the appearance of the skin and a slowing down of wrinkle formation is achieved.
The cosmetic compositions of the present invention are therefore particularly suitable for:
- slowing down ageing by maintaining an optimum fluidity of the skin cell membranes, a high membrane fluidity favouring internal intercellular exchanges and hence the optimum metabolism;
- improving the conditions of skins prematurely aged by the action of exogenous factors, via the above process.
Furthermore the cosmetic compositions according to the present invention are very well tolerated; they have no phototoxicity and their application to the skin for prolonged periods of time does not give rise to any systemic effect.
Another aspect of the present invention is, therefore, a method of treating the skin of a living human to improve the appearance of ageing skin, which method comprises applying to the skin a cosmetic preparation comprising a mixture of (a) "betaine", (as defined above) (b) ATP or an ATP generating system, (c) a magnesium salt, and (d) a potassium salt, in a cosmetically acceptable vehicle for topical administration. The mixture is contained in the cosmetic preparation in an amount effective to improve the appearance of ageing skin.
The following examples are merely illustrative of the scope of the present invention and are not intended as a limitation to the scope thereof.
For the sake of simplification, certain constituents of the compositions have been indicated by their tradenames or abbreviations whose meanings are reported hereinbelow.

~A

VITACELL LS1917 yeast extract manufactured by Laboratoires Serobiologiques S.A.
REVITALINE bovine spleen extract marketed by Pentapharm 5 OXYDERMINE bovine spleen extract marketed by Sederma SOLUTOL HS 15 polyethylene glycol 600 12-hydroxy stearate, marketed by BASF
CETIOL HE polyethylene glycol-7 glyceryl cocoate marketed by Henkel LABRAFAC
HYDROPHYLE polyethoxylated triglycerides containing 7-8 carbon atoms, marketed by Gattefosse 15 ABIL* 8851 B dimethycone copolyol marketed by Goldschmidt CARBOPOL* 934 I carboxypolymethylenes marketed by CARBOPOL 940 I Goldschmidt TWEEN* 60 ethoxylated sorbitan monostearate 20 TWEEN 20 ethoxylated sorbitan laurate EDTA ethylenediaminotetraacetic acid W B FILTER 2-ethylhexyl 4-methoxycinnamate (trademark PARSOL MCX) Example 1 Preparation to be reconstituted before use A) Solvent Carboxymethylcellulose 0.30 g Preservatives in propylene glycol 5.00 g Phenoxyethanol 0.5 g Methyl 4-hydroxybenzoate 0.1 g Ethyl 4-hydroxybenzoate 0.1 g Propyl 4-hydroxybenzoate 0.1 g Butyl 4-hydroxybenzoate 0.1 g Propylene glycol 4.1 g Ethoxylated hydrogenated castor oil 1.00 g Fragrance 0.20 g Demineralized water q.s. to 100.00 g * - Trade-marks ~A~

8a B) Powder Betaine 0.10 g Magnesium chloride 0.05 g Potassium chloride 0.10 g REVITALINE 0.50 g Lactose q.s. to 100.00 g Example 2 Protective day cream Betaine 0.05 g Magnesium chloride 0.05 g Potassium chloride 0.10 g REVITALINE 0.50 g TWEEN 60 2.60 g Silicon oil 1.00 g Cetyl alcohol 2.00 g Mineral oil 3.00 g Lanolin alcohol 1.00 g -Perhydrosqualene 1.00 g Sorbitan monostearate 2.40 g Cetyl palmitate 3.00 g Isopropyl palmitate 4.00 g UVB FILTER 2.00 g Tetrasodium EDTA 0.10 g CARBOPOL 934 0.30 g Triethanolamine 0.30 g Butylhyd~oxyLoluene 0.01 g Preservatives in butylene glycol 5.00 g Phenoxyethanol 0.5 g Methyl 4-hydLvxybenzoate 0.1 g Ethyl 4-hydLvxyLenzoate 0.1 g Propyl 4-hydLvxy~enzoate 0.1 g Butyl 4-hydroxyhen7O~te 0.1 g Butylene glycol 4.1 g Fragrance 0.30 g Demineralized waterq.s. to 100.00 g Example 3 Microemulsion Betaine 0.10 g Magnesium chloride0.05 g Potassium pyroglutamate 0.10 g REVITALINE 0.50 g TWEEN 60 20.00 g Glycerol 28.00 g Propylene glycol dipelargonate 40.00 g Ethyl 4-hydLoxyLenzoate0.30 g Fragrance 0.30 g ~ ;neralized water q.s. to 100.00 g 20172&S

-The microemulsion is obtained by mixing all the ingredients together and stirring until a clear solution is obtained.
Example 4 Microemulsion Betaine 0.20 g Magnesium gluconate 0.50 g Potassium gluconate 0.50 g OxYv~KMINE 0.50 g SOLUTOL HS 15 1.00 g LABRAFAC HYDROPHYLE 0.25 g CETIOL HE 0.20 g ABIL 8851 B 0.05 g Propylene glycol 12.50 g Ethanol 12.50 g CaL~uxy~olyvinyl polymer 0.40 g Fragrance 0.30 g Preservatives 0.30 g Colorant q.s.
Demineralized water q.s. to 100.00 g Triethanolamine q.s. to pH = 6 The microemulsion is prepared as described in Example 3.
Example 5 Fluid make-up foundation Betaine 0.10 g Magnesium gluconate 0.90 g Potassium gluco~Ate 0.60 g REVITALINE 0.50 g Ethoxylated soja sterols 4.00 g Soja sterols 0.50 g Glycerol monostearate 1.00 g Vegetable oil 1.50 g 2-Ethylhexyl palmitate 4.00 g Cetyl alcohol 0.50 g Capric/caprylic triglycerides 1.50 g Silicon oil 1.00 g Mineral oil 1.80 g Lanolin alcohols 0.20 g Propylene glycol dipelargonate 3.00 g Lecithin 1.00 g Preservatives in butylene glycol 5.00 g Phenoxyethanol 0.5 g Methyl 4-hydLoxybP~o~te 0.1 g Ethyl 4-hydroxyhPn7-o~te 0.1 g Propyl 4-hydroxybenzoate 0.1 g Butyl 4-hydroxybenzoate 0.1 g Butylene glycol 4.1 g CARBOPOL 940 0.20 g Triethanolamine 0.20 g Tetrasodium EDTA 0.10 g Butylhydroxytoluene 0.01 g Fragrance 0.30 g Example 6 Night cream Betaine 0.05 g Magnesium gluconate 0.90 g Potassium pyroglutamate 0.60 g REVITALINE 0.50 g Cetyl alcohol 2.00 g Stearin 2.50 g Glycerol monostearate 5.00 g Isopropyl palmitate 5.00 g Vegetable oil 3.00 g Mineral oil 2.00 g Perhydrosqualene 2.00 g Silicon oil 1.00 g Shea butter 2.00 g Preservatives in butylene glycol 5.00 g Phenoxyethanol 0.5 g Methyl 4-hydr~xybenzoate 0.1 g Ethyl 4-hydL~xyLenzoate 0.1 g Propyl 4-hyd~oxyLenzoate 0.1 g Butyl 4-hydLoxybenzoate 0.1 g 201726~
.

Butylene glycol 4.1 g Triethanolamine 5.50 g Tetr~s~; EDTA 0.10 g Fragrance 0.30 g Water q.s. to 100.00 g Example 7 Gel Betaine 0.10 g Magnesium gluconate 0.90 g Potassium gluconate 0.60 g REVITALINE 0.50 g CARBOPOL 940 0.20 g Polyethylene glycol 3.00 g Preservatives in butylene glycol 5.00 g Phenoxyethanol 0.5 g Methyl 4-hydLu~ybenzoate 0.1 g Ethyl 4-hydL~ybenzoate 0.1 g Propyl 4-hydL~xyLenzoate 0.1 g Butyl 4-hyd~xybenzoate 0.1 g Butylene glycol 4.1 g TWEEN 20 0.50 g Triethanolamine 0.25 g Fragrance 0.20 g D~ ;n~ralized waterq.s. to 100.00 g Example 8 Serum Betaine 0.10 g Magnesium gluconate0.90 g Potassium gluconate0.60 g Yeast extract (VITA OE LL LS 1917) 0.50 g HydLvxy~ropylmethylcellulose 0.30 g Propylene glycol S.00 g Glycerin 5-00 g `'Ethoxylated oleic alcohol 0.50 g Fragrance 0.30 g Tetrasodium EDTA 0.10 g ~ 2017265 Preservatives 0.50 g Bovine albumin 5.00 g Demineralized waterq.s. to 100.00 g Example 9 Make-up foundation Betaine 0.10 g Magnesium gluconate0.90 g Potassium pyroglutamate0.60 g Yeast extract (VITACELL LS 1917) 0.50 g Tetrasodium EDTA 0.10 g Carboxymethylcellulose0.20 g Aluminum magnesium silicate 1.00 g Ethoxylated sorbitan laurate 1.00 g Propylene glycol 5.00 g Titanium oxide 2.00 g Talc 1.00 g Pigments 1. ao g Triethanolamine -a . 50 g Preservatives 0.50 g Cetyl alcohol 1.00 g Lanolin alcohol 3.00 g Stearic acid 1.80 g Propylene glycol monostearate 3.00 g Isopropyl palmitate8.00 g Vegetable oil 2.00 g Antioxidants 0.05 g Fragrance 0.30 g Demineralized waterq~s. to 100.00 g

Claims (16)

1. A cosmetic composition which comprises a mixture of (a) betaine ((CH3)3N+CH2-COO-) or hydrated betaine or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) ATP or an ATP generating system, (c) a magnesium salt, and (d) a potassium salt, in a cosmetically acceptable vehicle for topical administration, said mixture being contained in said composition in an amount effective to improve the appearance of ageing skin.

2. The composition of claim 1 which comprises (a) from 0.001 to 1% of betaine ((CH3)3N+CH2-COO-) or hydrated betaine or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.045 to 4.5% of ATP or an ATP generating system, (c) from 10 ppb to 0.3% of Mg++, and (d) from 0.0001 to 0.6% of K+, the above percentages being by weight.

3. The composition of claim 2 which comprises (a) from 0.03 to 0.3% of betaine ((CH3)3N+CH2-COO-) or hydrated betaine or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.15 to 1.5% of ATP or an ATP generating system, (c) from 15 ppb to 0.1% of Mg++, and (d) from 0.001 to 0.2 % of K+, the above percentages being by weight.

4. The composition of claim 3 which comprises (a) from 0.05 to 0.25 % of betaine ((CH3)3N+CH2-COO-) or hydrated betaine or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.25 to 1% of an ATP generating system, (c) from 20 ppb to 0.06 % of Mg++, and (d) from 0.002 to 0.1 % of K+, the above percentages being by weight.

5. The composition of claim 1 wherein the ATP
generating system is an extract derived from eucaryotic cells.

6. The composition of claim 5, wherein said ATP
generating system is an extract derived from yeast cells or bovine spleen cells.

7. The composition of claim 5, wherein the ATP
generating system is selected from the group consisting of (a) a purified yeast cell extract marketed under the tradename VITACELL LS 1917, comprising amino acids and yeast cellular proteins, (b) a purified bovine spleen extract marketed under the tradename OXYDERMINE, comprising spleen cell proteins, fats and carbohydrates, and (c) a purified bovine spleen extract marketed under the tradename REVITALINE, comprising spleen cell proteins and nutrients.

8. The composition of claim 1 wherein the magnesium and potassium salts are selected from the group consisting of the corresponding chlorides, gluconates and pyroglutamates.

9. A cosmetic composition as claimed in claim 1, consisting of the recited ingredients.

10. A method of treating the skin of a living human to improve the appearance of ageing skin, which comprises applying to the skin a cosmetic preparation comprising a mixture of (a) betaine ((CH3)3N+CH2-COO-) or hydrated betaine or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) ATP or an ATP generating system, (c) a magnesium salt, and (d) a potassium salt, in a cosmetically acceptable vehicle for topical administration.

11. A method according to claim 10, wherein said ATP
generating system is an extract derived from yeast cells or bovine spleen cells.

12. A method according to claim 10, wherein said mixture is comprised of (a) from 0.0001 to 1% of betaine ((CH3)3N+CH2COO-) or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.045 to 4.5% of ATP or an ATP generating system, (c) from 10 ppb to 0.3% of Mg++, and (d) from 0.0001 to 0.6% of K+, the above percentages being by weight.

13. A method according to claim 10, wherein said mixture is comprised of (a) from 0.03 to 0.3% of betaine ((CH3)3N+CH2COO-) or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.15 to 1.5% of ATP or an ATP generating system, (c) from 15 ppb to 0.1% of Mg++, and (d) from 0.001 to 0.2% of K+, the above percentages being by weight.

14. A method according to claim 10, wherein said mixture is comprised of (a) from 0.05 to 0.25% of betaine ((CH3)3N+CH2COO-) or an addition salt or hydrated addition salt thereof with a dermocompatible acid, (b) from 0.25 to 1.0% of ATP or an ATP generating system, (c) from 20 ppb to 0.06% of Mg++, and (d) from 0.002 to 0.1% of K+, the above percentages being by weight.

15. A method according to claim 10, wherein said ATP
generating system is selected from the group consisting of (a) a purified yeast cell extract marketed under the tradename VITACELL LS 1917, comprising amino acids and yeast cellular proteins, (b) a purified bovine spleen extract marketed under the tradename OXYDERMINE, comprising spleen cell proteins, fats and carbohydrates, and (c) a purified bovine spleen extract marketed under the tradename REVITALINE, comprising spleen cell proteins and nutrients.

16. A method according to claim 10, wherein said magnesium and potassium salts are selected from the group consisting of the corresponding chlorides, gluconates and pyroglutimates.